Vol.
176,
May
15, 1991
No.
BIOCHEMICAL
3, 1991
AND
RESEARCH
BIOPHYSICAL
COMMUNICATIONS Pages
1062-1067
MOLECULAR CLONINGOF cDNA ENCODING THE 16 KDa SUBUNITOF VACUOLARHt-ATPase FROMMOUSECEREBELLUM Hironori
Hanada, Masahisa Hasebe, Yoshinori Moriyama, Masatomo Maeda, and Masamitsu Futai
Department of Organic Chemistry and Biochemistry, Institute of Scientific and Industrial Research, Osaka University, 8-l Mihogaoka, Ibaraki; Osaka 567, Japan Received April
1, 1991
cDNA for the 16 kDa subunit of vacuolar Ht-ATPase was cloned from mouse cerebellum and sequenced. The deduced polypeptide (155 amino acid residues; molecular weight, 15,808) was highly hydrophobic and homologous to the subunits of bovine adrenal medulla, Torpedo marmorata electric lobe, Drosophila and yeast. Glu-139 (supposed to be essential for proton transport) was also conserved as the potential dicyclohexylcarbodiimide binding site. The subunit had four transmembrane segments: Segment II and IV were highly homologous and Glu-139 was located in Segment IV. The roles of the non-conserved regions are e 1991Academic Press,Inc. discussed.
Vacuolar H+-ATPase (V-ATPase) has been found in endomembranesystems of eukaryotic gradients
cells
and shown to translocate
that are essential
for organellar
hormones and neurotransmitters Its function hydrolases similar
in acidifying (2).
structure
functions
such as accumulation of
granules (for review,
V-ATPase is unable to synthesize
to FoF1 ATPase (ATP synthase) and consists The peripheral
sectors.
hydrolysis
and a similar
complicated
sector
has a catalytic
subunit
structure
membranesector consists of at least 2 different
and 20 kDa, and acts as a proton pathway. to the membrane sector
Abbreviations
and inhibits
1062
1).
for lysosomal ATP, it
has a
of membraneand site(s)
to that
for
of F1.
ATP The
subunits of 16 kDa
The 16 kDa subunit
used: V-ATPase, vacuolar H’-ATPase; DCCD, dicyclohexylcarbodiimide.
0 1991 by Academic Press, Inc. in any form reserved.
of reproduction
see ref.
Baf ilomycin A1 specifically
ATPase (3).
0006-291X/91$1.50 Copyright All rights
to form electrochemical
lysosomes provides optimal conditions
Although
peripheral
intrinsic
in secretory
protons
binds of V-
Vol.
176,
No.
3, 1991
BIOCHEMICAL
ATPase and the c subunit a common ancestral cation
AND
of F,Fl
protein
RESEARCH
ATPase have been suggested
(4),
of the 16 kDa subunit
BIOPHYSICAL
COMMUNICATIONS
to be derived
and as in the case of the c subunit, with
dicyclohexylcarbodiimide
from
modifi-
(DCCD) inhibits
ATPase activity. Recently,
Moriyama
constituent
and Futai
of the synaptic
membrane protein).
granules,
of neurotransmitters.
ATPases of these other
vesicles
organelles
different
of this
problem,
which
to focus
study,
mouse cerebellum.
roles
to those
marmorata
electric
lobe
homologies
to
latter
the
is
(8)) two
pump for
to the V-ATPases and lysosomes,
in different
Drosophila
(9),
subunits
were
of
this
adrenal
and the role
found
in
or whether For
protein
on
in
the
V-ATPase
(6,
7).
the 16 kDa subunit
of
subunit
showed
medulla
(4),
and yeast lower
V-
subunit
of the entire for
the
organelles.
intrinsic
a cDNA coding
from bovine
accu-
to know whether
a major
sequence
of the subunit
properties
seems to be a suitable
in assembly
acid
of the subunits
The topology
it
and sequenced
The amino
homology
granules
a major
20 % of the total
as a primary
interest
are expressed
because
is
the same enzymatic
similar
the 16 kDa subunit
attention,
we cloned
of
as chromaffin
membrane and has essential In this
is
V-ATPase
about
and functions It
of V-ATPase
that
(constituting
are structurally
such
isoforms
study
formers.
vesicles
reported
This ATPase has essentially
as V-ATPase of chromaffin mulation
(5)
than
(lo), those
Torpedo
although to
of non-conserved
high
the
its two
regions
are discussed. MATERIALS AND METHODS A mouse (ICR) cerebellum cDNA library constructed with bacteriophage Phage plaques were transkgtll was propagated in E. coli Y1090 (r-e&) (11). ferred to nitrocellulose filters (Schleicher and Schiill) and hybridized with an equimolar mixture of four 65-base synthetic oligonucleotides corresponding to the antisense (positions 367-431 and 478542) and sense (positions 422-486 and 533-597) strands of the carboxyl terminal half of the bovine 16 kDa subunit (4). Ten positive clones were isolated from 250,000 plaques. Although they were classified into four groups (I, hmvpl, 3, 5, 128; II, ;imvp8, 20, maps, portions of their se163; III, ~mvp4; VI, Amvp2) from the restriction The cDNAs encoding the amino terminal region quences overlapped (Fig. 1). were amplified from the library by the polymerase chain reaction with Taq DNA polymerase (Perkin Elmer Cetus) using 24-base primers corresponding to the 1gtll left arm (GGTGGCGACGACTCCTGGAGCCCG) and antisense strand (position 328The cDNAs were subcloned into pBluescript II SK M13(+) and both 341, Fig. 1). strands were sequenced (12). 1063
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase and the fragment of E. coli DNA polymerase I were purchased from Takara Shuzo (Osaka, Jap3az) and Nippon Gene (Toyama, Japan). (Kyoto, Japan), Toyobo PlATP (5,000 Ci/mmol) were purchased [,-32P)dCTP (3,000 Ci/mmol)and [7from AmershamCorp. Sequenase was from United State Biochem. Corp. A random priming DNA labeling kit was from Boeringer-Mannheim. All other chemicals used were the highest grade commercially available. large
RESULTSANDDISCUSSION The nucleotide subunit
of V-ATPase
fragments amplified tional
initiation
sequence of the cDNA encoding the was determined
using
hybridization-positive
by the polymerase chain reaction site
was assigned to the Met-l
sequences around the initiation
found between the in-frame termination (Fig.
2).
subunit
A termination
was found next
the
polyA
tail
(Fig
signal
1).
(4) and its
codon (13).
and
The transla-
similarity
to
No Met codon was
-135) and Met-l
the reading
to the Lys-155 codon.
(557 bp), there was a potential
(Fig.
codon (position
codon (TAG) for
clones
codon from the homology of
the sequence to that of the bovine adrenal subunit the eukaryotic
cerebellum 16 kDa
mouse
codon
frame of the 16 kDa
In the 3’-noncoding
for polyadenylation
region
(14) upstream of
2).
Pairwise comparisons of the nucleotide
sequences of the
16 kDa subunit with those of bovine adrenal medulla (4),
mouse
cerebellum
the Torpedo marmorata
100 bp mvp2
f >
-
1